Nitric oxide (NO), a gaseous free radical, was once considered mainly as an environmental pollutant from car exhausts and in city smog and cigarette smoke. This view toward nitric oxide was changed in 1987, the year in which NO was discovered to be produced in humans (see, for example, Ignarro et al., in Proc. Natl. Acad. Sci., USA, 84:9265-69 (1987) and Palmer et al., in Nature, 327:524-26 (1987)). First identified as an endothelium-derived relaxation factor, NO is now recognized as a new kind of cell signaling molecule that regulates the functions of many mammalian cells and tissues.
NO is generated by the enzymatic cleavage of L-arginine, catalyzed by the nitric oxide synthase enzyme (NOS; see, for example, Rodeberg et al., in Am. J. Surg., 170:292-303 (1995), and Bredt and Snyder in Ann. Rev. Biochem., 63:175-95 (1994)). Two major forms of NOS, constitutive and inducible enzymes, have been identified. Under physiological conditions, a low output of NO is produced by the constitutive, calcium-dependent NOS isoform (cNOS), which is present in numerous cells, including endothelium and neurons. This low level of nitric oxide is involved in a variety of regulatory processes, e.g., blood vessel homeostasis, neuronal communication and immune system functions. On the other hand, under pathophysiological conditions, a high output of NO is produced by the inducible, calcium-independent NOS isoform (iNOS), which is expressed in numerous cell types, including endothelial cells, smooth muscle cells and macrophages. These high levels of nitric oxide have been shown to be associated with many inflammatory and infectious diseases and conditions, such as septic shock, over expression of cytokines, diabetes, allograft rejection, inflammatory bowel disease, etc.
Nitric oxide is a potent vasodilator (see, for example, Palmer in Arch. Surg., 128:396-401 (1993) and Radomski & Moncada in Thromb. Haemos., 70:36-41 (1993)). For example, in blood, NO produced by the endothelium diffuses isotropically in all directions into adjacent tissues. As NO diffuses into the vascular smooth muscle, it binds to guanylate cyclase enzyme, which catalyzes the production of cGMP, inducing vasodilation (see, for example, Ignarro, L. J., Ann. Rev. Toxicol. 30:535-560 (1990); Moncada, S., Acta Physiol. Scand., 145:201-227 (1992); and Lowenstein and Snyder, Cell, 70:705-707 (1992)).
The overproduction of nitric oxide causes an extreme drop in blood pressure, resulting in insufficient tissue perfusion and organ failure, syndromes that are associated with many diseases and/or conditions (e.g., septic shock, stroke, over expression of cytokines, allograft rejection, and the like). The overproduction of nitric oxide is triggered by a number of stimuli, such as, the overproduction of inflammatory cytokines (e.g., the overproduction of interleukin-1, interferons, endotoxin, and the like). Additionally, the overproduction of NO has been found to be one of the major side-effects of cytokine therapy (see, for example, Miles et al., in Eur. J. Clin. Invest., 24:287-290 (1994) and Hibbs et al., in J. Clin. Invest., 89:867-877 (1992)). Thus, abnormally elevated nitric oxide levels have been associated with many inflammatory and infectious diseases.
The half-life of NO in vivo is only 3-5 seconds, a short lifetime that makes it very difficult to detect and quantify. Several biophysical techniques have been developed for the measurement of NO levels in aqueous solution. These include chemiluminescence assay (see, for example, Downes et al., Analyst, 101:742-748 (1976)), oxyhemoglobin assay (see, for example, Kelm and Schrader, Cir. Res., 66:1561-1575 (1990)), GC-MS detection (see, for example, Palmer et al., Nature (London), 327:524-526 (1987)), and nitrosyl-hemoglobin formation detected by electron paramagnetic resonance (EPR) spectroscopy at liquid nitrogen temperature (see, for example, Lancaster et al., Proc. Natl. Acad. Sci. USA, 87:1223:1227 (1990)).
Production of NO can also be indirectly detected by measuring its end products, NO.sub.2.sup.- /NO.sub.3.sup.- (see, for example, Palmer et al., supra). None of these techniques in their present forms, however, can be used for in vivo detection of NO production. Recently, an invasive electrochemical microsensor to detect NO levels in blood vessels of healthy human volunteers has been described (see, for example, Vallance et al., Lancet, 346:153-154 (1995)).
Dithiocarbamates are a class of low molecular-weight sulphur-containing compounds that are effective chelators (see, for example, Shinobu et al., Acta Pharmacol et Toxicol., 54:189-194 (1984)). For example, diethyldithiocarbamate (DETC) is used clinically for the treatment of nickel poisoning. Recently, it was found that N-methyl-D-glucamine dithiocarbamate (MGD) chelates with ferrous iron as a two-to-one (MGD).sub.2 -Fe! complex, which in turn interacts strongly with NO, forming a stable and water-soluble complex in aqueous solution, i.e., (MGD).sub.2 -Fe-NO! (see, for example, Lai & Komarov, FEBS Lett., 345:120-124 (1994)). The latter complex gives rise to a sharp three-line spectrum with g.sub.iso =2.04, characteristic of a nitrosyl-Fe-dithiocarbamate complex which can readily be detected by EPR spectroscopy at ambient temperatures. This method of detecting NO in body fluids in real time has recently been described by Lai in U.S. Pat. No. 5,358,703.
There is, however, still a need in the art for more rapid, preferably non-invasive methods for the detection of nitric oxide in body fluids.